Medical Immunology BioMed Central

Review Open Access

The quantal theory of how the immune system discriminates
between "self and non-self"
Kendall A Smith*

Address: The Division of Immunology, Department of Medicine, Weill Medical College, Cornell University, New York, New York, United States
of America
Email: Kendall A Smith* - kasmith@med.cornell.edu
* Corresponding author

Published: 17 December 2004 Received: 12 December 2004

Accepted: 17 December 2004
Medical Immunology 2004, 3:3 doi:10.1186/1476-9433-3-3
This article is available from: http://www.medimmunol.com/content/3/1/3
© 2004 Smith; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract
In the past 50 years, immunologists have accumulated an amazing amount of information as to how
the immune system functions. However, one of the most fundamental aspects of immunity, how
the immune system discriminates between self vs. non-self, still remains an enigma. Any attempt to
explain this most intriguing and fundamental characteristic must account for this decision at the
level of the whole immune system, but as well, at the level of the individual cells making up the
immune system. Moreover, it must provide for a molecular explanation as to how and why the cells
behave as they do. The "Quantal Theory", proposed herein, is based upon the "Clonal Selection
Theory", first proposed by Sir McFarland Burnet in 1955, in which he explained the remarkable
specificity as well as diversity of recognition of everything foreign in the environment. The "Quantal
Theory" is built upon Burnet's premise that after antigen selection of cell clones, a proliferative
expansion of the selected cells ensues. Furthermore, it is derived from experiments which indicate
that the proliferation of antigen-selected cell clones is determined by a quantal, "all-or-none",
decision promulgated by a critical number of cellular receptors triggered by the T Cell Growth
Factor (TCGF), interleukin 2 (IL2). An extraordinary number of experiments reported especially
in the past 20 years, and detailed herein, indicate that the T cell Antigen Receptor (TCR) behaves
similarly, and also that there are several critical numbers of triggered TCRs that determine different
fates of the T cells. Moreover, the fates of the cells appear ultimately to be determined by the TCR
triggering of the IL2 and IL2 receptor (IL2R) genes, which are also expressed in a very quantal
fashion. The "Quantal Theory" states that the fundamental decisions of the T cell immune
system are dependent upon the cells receiving a critical number of triggered TCRs and IL2Rs
and that the cells respond in an all-or-none fashion. The "Quantal Theory" accounts fully for
the development of T cells in the thymus, and such fundamental cellular fates as both "positive" and
"negative" selection, as well as the decision to differentiate into a "Regulatory T cell" (T-Reg). In the
periphery, the "Quantal Theory" accounts for the decision to proliferate or not in response to the
presence of an antigen, either non-self or self, or to differentiate into a T-Reg. Since the immune
system discriminates between self and non-self antigens by the accumulated number of triggered
TCRs and IL2Rs, therapeutic manipulation of the determinants of these quantal decisions should
permit new approaches to either enhance or dampen antigen-specific immune responses.

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Introduction
Perhaps one of the most unique and fundamental aspects
of the immune system is the clonal nature of the response
to the introduction of antigen. The "Clonal Selection The-
ory" as originally formulated by Burnet stated that the
immune system is made up of cells, each of which are
only capable of reacting with a single antigenic mole-
cule[1]. Thus, Burnet improved upon the "Natural-selec-
tion theory of antibody formation" proposed by Neils
Jerne[2], by giving the immune response a cellular basis.
Also, Burnet introduced the notion that after antigen
selection, the reactive cell clones proliferate, and the
resulting expanded population of cells would only then
be capable of removing the offending foreign antigen.
Consequently, one of the most crucial decisions required
of an antigen-selected cell is whether to undergo cell cycle
progression. Ultimately this decision determines one of
the other most fundamental characteristics of the immune
system, the ability to discriminate between self vs. non-
self.
At the time that Burnet formulated the concept of clonal
selection in the mid 1950s, the identity of the cells com-
prising the immune system was unknown. Plasma cells
had been found to be the source of antibodies[3], but
lymphocytes had not yet been identified as the precursors
of plasma cells. Moreover, thymic-derived lymphocytes (T
cells) and bone marrow-derived lymphocytes (B cells)
were not to be discovered for almost two more decades [4-
6].
In the intervening 50 years since Burnet formulated his
theory, much has been discovered and even more has
been proposed to explain how the immune system func-
tions, especially how the discrimination between self vs.
non-self is made. Since 1959, several modifications of
Burnet's original model have been offered, each of which
introduced additional cells in an attempt to explain how
the entire system could react with absolutely everything in
the environment, but not react with any molecules com-
prising self [7-15]. However, none of the models pro-
posed thus far have focused on one of the most
fundamental aspects of the Clonal Selection Theory as
originally formulated by Burnet, i.e. the molecular forces
driving the proliferative expansion of the antigen-selected
cell clones.
We now know that lymphocytes make up the immune
system, and we know the molecular structures of the anti-
gen receptors expressed by both T cells and B cells. Conse-
quently we know that Burnet was largely correct. Each cell
expresses a unique antigen receptor that has the capacity
to bind only a single antigenic epitope. The exception to
this rule is the "allelic inclusion" of two T Cell Receptor
(TCR) α-chains, resulting in approximately 30% of α/β-
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chain bearing T cells as having dual antigenic specificity.
As well, it is known that T cells recognize epitopes com-
prised of short peptides of only a few amino acid residues
bound to molecules encoded by the Major Histocompati-
bility gene Complex (MHC), while B cells recognize both
the tertiary surface structure of larger molecules, as well as
linear determinants of molecules. Despite this informa-
tion, it still remains unknown how the immune system
discriminates between non-self vs. self-molecules, in that
the molecular nature of the T cell Antigen Receptors (TCR)
and the B cell Antigen Receptors (BCR) that recognize
both self- and non-self- epitopes are identical. Moreover,
the molecular natures of self-epitopes and non-self -
epitopes are identical. Accordingly, it is even more per-
plexing as to how the immune system manages this
discrimination.
One key to understanding the way in which the immune
system operates is the observation that the capacity to rec-
ognize and respond to antigen is dependent on the dose
of antigen introduced[16]. Thus, there appears to be sev-
eral outcomes possible, such that at low antigen doses
there is no detectable response, and with increasing doses
of antigen there may be the induction of tolerance, while
even higher doses are necessary to trigger an immune
response, which involves more and more antigen-reactive
cells as the antigen dose increases. At very high antigen
doses there may even be a "paralysis" induced.
Another key to understanding the immune response
resides in the realization that the absolute frequency of
potential antigen-reactive lymphocytes is very low before
the introduction of antigen, on the order of 1 in a million
cells up to 1 in 10,000 cells (i.e. 10-6 to 10-4). However,
after antigen selection, proliferative clonal expansion
increases the frequency of antigen-reactive cells to as high
as 1 in 10 cells, an astonishing 1–100,000-fold increase
[17-20]. Thus, the decision by individual cells to prolifer-
ate in response to recognition of an antigen is the critical
decision at the cellular level that controls the ability of the
whole immune system to discriminate between self vs.
non-self.
Until the discovery of mitogenic cytokines, it was assumed
that antigens are solely responsible for stimulating prolif-
eration. It is now known that there are antigen-activated
cytokines with T cell Growth Factor (TCGF) activity that
provide molecular signals that markedly stimulate cell
cycle progression. Since the principle cytokine with TCGF
activity driving T cell proliferation is the interleukin-2
(IL2) molecule[21,22], the molecular mechanism
whereby IL2 promotes cell cycle progression is of utmost
importance.
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Figure
The IL2 1binding and biological response curves are coincident
The IL2 binding and biological response curves are coinci-
dent. Radiolabeled IL2 and purified homogeneous IL2 were
used in parallel experiments with the same IL2R+ T cell pop-
ulation to determine the relationship between IL2 binding
and IL2-promoted T cell proliferation as monitored by 3H-
TdR incorporation. From reference 25.
Accordingly, to understand how the immune system dis-
criminates between self vs. non-self antigens, the IL2 mol-
ecule is first examined, particularly how IL2 promotes the
proliferation of antigen-selected cells. Then, the cellular
and molecular determinants of IL2 production and IL2R
expression are traced. Ultimately, the maturation of T cells
in the thymus must be considered, to understand how
cells determine whether to produce IL2 or not, and
whether to respond to it or not. Most of the discussion
that follows is focused on T cells, but the general princi-
ples developed apply to B cells as well.
The Quantal Nature of IL2-Promoted T Cell
Proliferation
At the level of the individual cell, the proliferative
response to IL2 is quantal, i.e. it is "all-or-none"[23,24].
However, to proceed beyond simply a description of this
phenomenon, one must understand the molecular basis
for the response of individual cells to IL2, so as to predict
the behavior of the immune system as a whole.
Soon after the development of the radiolabeled-IL2 bind-
ing assay[25], which permitted IL2 receptors to be quanti-
fied and defined for the first time, experiments could be
performed to ascertain how the concentrations of IL2 that
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bind to IL2Rs compare with the IL2 concentrations that
promote T cell proliferation. It was revealed that the bind-
ing and biological response curves are coincident, as
shown in Figure 1[25]. The IL2 concentration-dependent
response ranges from 1–100 pM, and the 50% effective
concentration (EC50) equals the equilibrium dissociation
constant (Kd) of the IL2-IL2R interaction, both of which
are ~ 10 pM. Although this is a very high affinity for a lig-
and-receptor interaction (e.g. most TCR-peptide-MHC
affinities are ~ a million-fold lower), it is noteworthy that
in molecular terms, the EC50/Kd = 6 billion molecules/
mL.
Of course, these experiments were performed using cell
populations, and symmetrically sigmoid log-dose response
curves are quite familiar in these circumstances. However,
from these experiments it was not possible to discern
whether the low thymidine incorporation found at low
IL2 concentrations was due to all of the cells in population
incorporating only a little thymidine, or whether only a
few of the cells could respond at low IL2 concentrations.
Therefore, it was not until the IL2 dose-response relation-
ship could be examined at the single cell level did it
become apparent that the cell populations are comprised
of individual cells that differ markedly as to their respon-
siveness to the mitogenic ligand. Thus, as shown in Figure
2 using propidium iodide staining of DNA and the flow
cytometer to compare with thymidine incorporation, it
was found that some cells of an asynchronously prolifer-
ating population respond by proliferating to very low IL2
concentrations, e.g. only 1 pM, while others need 100-
fold higher IL2 concentrations. Moreover, the marked het-
erogeneity in IL2 responsiveness could not be explained
on a genetic basis, since even cloned cell populations
behaved in an identical fashion.
Once it is realized that the effective IL2 concentrations
span 2 orders of magnitude, the question becomes why
some cells are capable of responding at only 1 pM, while
others require IL2 concentrations that are 100-fold higher.
The only logical answer to this question is that there must
be intrinsic cellular differences, and that these differences
are manifest in molecules that are critical for signaling cell
cycle progression. Therefore, in experiments focused on
understanding how IL2 promotes T cell proliferation after
antigen activation, we found that in addition to the affin-
ity of the IL2/IL2R interaction, and the concentration of
IL2, the other variables involved are the IL2 receptor
(IL2R) density, and the duration that the IL2 and the IL2R
molecules interact[26].
In order to design experiments to approach these varia-
bles, it was necessary to use cell populations that were syn-
chronized in early G1, so as to follow the rate at which
individual cells progressed through G1 into S-phase. Thus,
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responsiveness
The
at
Figure
theIL2single
2biological
cell level
dose-response
by a markedrelationship
heterogeneity
is determined
of
The IL2 biological dose-response relationship is determined
at the single cell level by a marked heterogeneity of respon-
siveness. Asynchronously proliferating IL2R+ cells were
exposed to varying concentrations of IL2 for 18 hours. Sub-
sequently, cell aliquots were either pulsed for 4 hours with
3H-TdR, or stained with propidium iodide prior to single cell
analysis by flow cytometry. The cell population incorporates
3H-TdR in an IL2 concentration dependent manner, and the
amount of 3H-TdR incorporation at each IL2 concentration is
determined by the absolute number of cells that have
entered S-phase, as indicated by the single cell analysis by
propidium iodide staining. From reference 26.
if G0/1 synchronized IL2R+ cells are exposed to two differ-
ent IL2 concentrations, one receptor saturating and
another only half-saturating; the receptor-saturating con-
centration promotes cell cycle progression twice as rapidly
as the half-saturating IL2 concentration. However, as the
ligand concentration dependency of cell cycle progression
of cell populations was well known, these results were not
surprising.
However, it was surprising to find that if one exposes two
cell populations, one with a higher IL2R density than
another, to the same receptor saturating IL2 concentra-
tion, the cell population with the higher IL2R density
traverses G1 and enters S-phase more rapidly than the
population with the lower IL2R density (Figure 3).
These observations predict that the basis for the character-
istic sigmoid IL2 log-dose response curve (Figure 1) is
only explicable because of heterogeneity of IL2R density
within a given population of T cells. The heterogeneity of
IL2R density/cell is readily appreciated from examination
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progression
The
Figure
effect
3 of IL2R density on the rate of T cell cycle
The effect of IL2R density on the rate of T cell cycle progres-
sion. Two G0/1 synchronized T cell populations that differed
3-fold in mean IL2R density were exposed to an IL2R saturat-
ing concentration (250 pM) for 48 hours and 3H-TdR incor-
poration was monitored as indicated at 1 hour intervals. The
cells with the higher IL2R density (solid circles) entered S-
phase before the cell population with the lower IL2R density
(solid triangles). From reference 26.
of the flow cytometry plot of the log-normal distribution
of IL2Rs as shown in Figure 4. Thus, at low IL2 concentra-
tions, i.e. ~ 1 pM, only cells with the highest IL2R density
are capable of responding. As the cell population is
exposed to increasingly higher IL2 concentrations, cells
with lower IL2R densities will meet the quantal require-
ment to enter the cell cycle. Finally, as the IL2 concentra-
tion reaches levels that saturate all IL2Rs, even cells with
the lowest IL2R density can reach the critical number.
These findings indicate that cells reach a decision to
undergo cell cycle progression based on some critical
number of IL2-IL2R intermolecular reactions at the cell
surface. Also, they indicate that the duration of the IL2-
IL2R interaction plays a role, such that it appears that if a
cell has a low density of IL2Rs, the critical number of IL2-
IL2R interactions can still be reached, but a longer time
interval is necessary. Also, the data indicate that if one
interrupts the IL2-IL2R interaction before the critical
number of interactions is attained, then cell cycle
progression will not occur, as shown for a 3-hour expo-
sure in Figure 5. In other words, the cell "counts" the
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cytometry
IL2R
Figuredensity
4 determined at the single cell level by flow
IL2R density determined at the single cell level by flow
cytometry. IL2R+ T cells were labeled with anti-Tac (CD25)
monoclonal antibody and analyzed by single cell flow cytome-
try. The IL2R density varies among cells within he population
over 3 orders of magnitude. From reference 26.
number of triggered receptors and waits until the requisite
number has accumulated before proceeding beyond G1 to
S-phase[24,27].
IL2/IL2R binding is rapid and comes to steady state within
10 minutes[25,28], yet several hours of IL2 exposure are
necessary to reach the critical number of IL2/IL2R interac-
tions required to trigger cell cycle progression. Therefore,
it must be concluded that the requisite number of IL2Rs is
not present on any cells before the addition of IL2.
Instead, new receptors must be continuously synthesized,
expressed on the cell surface and serially engaged over sev-
eral hours to finally reach the quantal number. Accord-
ingly, the decision to divide is regulated with exquisite
high fidelity, and the decision must be quantal. Otherwise
a cell might only partially replicate its DNA before
undergoing cytokinesis, a situation clearly incompatible
with life.
An estimate of this critical number of IL2/IL2R interac-
tions required can be calculated[24], knowing the initial
mean number of receptors (~ 750 Rs/cell), and the rate of
internalization and degradation of IL2 bound IL2Rs from
the cell surface at steady state (t1/2 = 15 minutes). Thus, the
rate constant, κ = ln2/15 min = 4.67 × 10-2 min-1, governs
the rate of new receptor synthesis necessary to maintain
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Figure
The effect
ative response
5 of varying
of G0/1the
synchronized
IL2 exposure
IL2R+
period
T cells
on the prolifer-
The effect of varying the IL2 exposure period on the prolifer-
ative response of G0/1 synchronized IL2R+ T cells. Aliquots of
synchronized cells were exposed to IL2 for varying intervals
(3, 6, 11, 26 hours) then washed and placed into culture
without IL2 and pulsed with 3H-TdR. Symbols: IL2 exposure,
3 hr (solid circles), 6 hr (open circles), 11 hr (solid triangles),
and 26 hr (open triangles). Inset, shows the 3H-TdR incorpo-
ration of each cell population in response to an IL2R saturat-
ing IL2 concentration (250 pM) monitored for 1 hr at the
times indicated. From reference 26.
the surface expression at steady state. Moreover, 11 hours
(660 minutes) of IL2 exposure is necessary to trigger 50%
of the cells within the population to undergo cell cycle
progression (Figure 5). Thus, the mean number of trig-
gered IL2Rs necessary is:
R# (R/cell) = κ × R# @ steady state
= 4.67 × 10-2 min-1 × 750 R/cell
= 35 R/cell/min × 660 min
= 23,100 triggered R/cell
It follows that if the mean number of IL2Rs at steady state
is lower, e.g. only 375 Rs/cell, 22 hours would be required
to reach the quantal number and if the rate of new recep-
tor synthesis is doubled, maintaining twice as many,
1,500 sites/cell, then only 5.5 hours would be required to
trigger the cells.
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Intracellular Sensors of the Extracellular Signals
There are 3 chains that make up the trimeric IL2R that has
an extremely high affinity for IL2, termed α (CD25)[29],
β (CD122)[30] and γ (CD132)[31]. Careful kinetic and
equilibrium binding experiments using radiolabeled IL2
revealed that the α chain contributes a very rapid
association rate (κ = 107 M-1sec-1), while the β chain con-
tributes a slow dissociation rate (κ' = 10-4 sec-1), which
together yield the high affinity (Kd = κ'/κ = 10-11 M) for
the ligand observed at steady state[28].
Recent experiments focused on the energetics of assembly
of IL2/IL2R signaling complexes have revealed that in
solution, the IL2R α and β chains bind to one another,
whereas the α chain does not bind to the γ chain[32].
Moreover, the γ chain can only bind to α,β dimers or iso-
lated β chains if IL2 has already bound to these receptor
chains. These data support the interpretation that the α,β
dimer probably is formed on the surface of antigen-acti-
vated T cells, and serves as the initial receptor complex
capable of binding IL2. Moreover, the 10–20-fold excess
expression of the α chain vs. the β chain favors the forma-
tion of the α,β heterodimer by the law of mass action.
Subsequently, the IL2, α,β trimeric complex then can
attract and bind the γ chain, forming a quaternary com-
plex that is capable of signaling the cell interior. These
energetic experiments provide an explanation for the IL2-
dependency of signaling, since signaling only occurs
when the γ-chain and the β chain are brought into close
proximity, thereby activating the tyrosine-specific kinases,
JAK 1 and 3, which are already bound to the β and γ chains
respectively[33]. Accordingly, the assembly and mainte-
nance of this energetically stable multicomponent macro-
molecular signaling complex is a fundamental
requirement for the cell ultimately to make the quantal
decision to divide.
The important downstream events in IL2-promoted T cell
cycle progression are the JAK-dependent activation of at
least two distinct proliferative signaling pathways. One is
mediated by the transcription factor STAT5, while the
other is mediated by the adapter molecule Shc, which acti-
vates phosphatidylinositol 3-kinase (PI3K). Both cyclin
D2 and D3 are expressed in response to IL2R trigger-
ing[34], and recent studies have shown that the STAT5
and PI3K pathways play distinct, but coordinated roles in
the quantal IL2R induction of progression through the
Restriction Point (R-point) in the G1 phase of the cell cycle
[35-37]. In addition to Shc, STAT5 also facilitates the acti-
vation of the PI3K pathway by a delayed mechanism that
requires protein synthesis, and PI3K activity is essential
for the induction of cyclin D2 expression by STAT5. PI3K
activity is required for the optimal binding of RNA
polymerase II to the promoters of cyclin D2 as well as
other IL2/STAT5-induced genes.
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Because of these findings, it has been proposed that the D
cyclins serve as intracellular sensors of the extracellular
signals [38] generated at the cell surface by the formation
of the stable quaternary IL2/IL2R signaling complex[39].
Thus, cyclin D2 and D3 complex with the cyclin-depend-
ent kinases (cdk) 4 & 6 and the p27 protein, thereby form-
ing active kinases that initiate phosphorylation of the Rb
proteins that repress the E2F transcription factors. The
E2Fs are already bound to response elements that regulate
the expression of multiple genes, the expression of each of
which is critical for both nucleotide synthesis and DNA
replication. Also, the E2F transcription factors regulate the
expression of multiple genes required for formation of the
Pre-replication Complexes (Pre-RCs), which must first
assemble and then disassemble at sites on DNA termed
Origins of Replication (Ori) before the DNA strands can
separate, thereby allowing DNA duplication. Accordingly,
the cyclin D/cdk/p27-dependent phosphorylation of Rb
has been proposed to initiate the passage through the R-
point, and has been described as a quantal molecular
"binary switch"[39,40].
However, recent experiments with mice that have had all
3 of the cyclin D genes deleted have revealed that
although hematopoiesis is dependent on the coordinated
expression of the cyclin D genes, nonhematopoietic cells
can proliferate in the absence of the D-type cyclins and
their cyclin-dependent kinases 4 and 6 (cdk4/6)[41,42].
Even so, mouse embryo fibroblasts lacking type D cyclins
proliferate more slowly to stimulation by serum by com-
parison to their wild type counterparts. Therefore, it
appears that there are at least 2 distinct pathways whereby
extracellular signals can trigger G1 progression, one
involving cyclin D and another that is cyclin D
independent.
At this time, it remains unknown as to whether T cells can
be stimulated to proliferate by both cyclin D-dependent
and independent pathways, or only by cyclin D-depend-
ent (and therefore IL2R/STAT5-dependant) pathways.
However, it is clear that T cells are in G0 until activated by
initial signals received via the TCR, so that it still remains
possible that T cells may be capable of proliferating in
response to both TCR- and IL2R-derived signals[33,43,44].
Alternatively, the TCR may be responsible for the G0 to G1
transition, while the IL2R is responsible for G1 progres-
sion to the R-point and S-phase transition. However,
another possibility remains that it still may well be that
TCR-derived signals can initiate early rounds of cell divi-
sion, but that for a fully developed clonal expansion, IL2/
STAT5-dependent signals are necessary. If so, one would
predict that TCR-mediated cell cycle progression is STAT5-
and cyclin D-independent, so that the role of IL2 is to
markedly accelerate and extend cell cycle progression ini-
tiated by the TCR.
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The Quantal Regulation of IL2 Gene Expression
Once one realizes that the quantal decision of T cells to
proliferate is based upon a critical number of IL2-IL2R
interactions, it becomes immediately obvious that the
availability of IL2, together with the extent of IL2R expres-
sion, ultimately determines whether a immune response
occurs that will be detectable at the systemic level. Since
the discovery [45-51] and elucidation of the nature of the
T Cell antigen Receptor (TCR) over the past 20 years [52-
56], data have accumulated indicating that the regulation
of IL2 gene expression and IL2R gene expression is under
a tight and complex cell surface signaling mechanism that
involves not only the TCR, but other surface molecules as
well.
Before the elucidation of the structure and function of the
receptors involved in antigen recognition, it was difficult
to envision how the IL2/IL2R system is regulated. How-
ever, the principles from the quantal IL2-IL2R signaling of
cell cycle progression can now be extrapolated to this
more complex signaling system, and herein resides the
ultimate control of "self-non-self" recognition and
response. Like the IL2R[24,27], the TCR also is capable of
counting the number of antigen interactions so as to
acquire the critical number of triggered receptors neces-
sary for IL2 gene expression.
Since the cloning and sequencing of the IL2 cDNA[57]
and gene[58], detailed studies have revealed the nature of
the molecules controlling IL2 expression. There are 3 dis-
tinct response elements in the promoter region of the IL2
gene that bind members of distinct families of transcrip-
tional activating factors [59-61]. These factors include
Activating Protein-1 (AP-1), Nuclear Factor of Activated T
cells (NF-AT), and the Nuclear Factor kappa B/Rel (NF-
κB).
Individual IL2 transcription factors from these three fam-
ilies cannot bind stably to their target DNA response ele-
ments in vivo without coengagement of each of the
distinct factors that bind at neighboring sites[62]. Also, if
the members of any one of these factors is prevented from
binding to the IL2 promoter region, there is a marked
attenuation of IL2 gene transcription[63]. Moreover, even
after the factors have bound, inactivation of any of the
three transcription factors pharmacologically extinguishes
the binding of all three factors, thereby aborting transcrip-
tion[64]. Therefore it has been proposed that there is a
nonhierarchical, cooperative enhancement of binding at
the IL2 gene locus, and that this binding and transcrip-
tional activation of IL2 gene expression is consequently,
quantal.
The quantal binding of IL2 transcription factors to the IL2
enhancer and promotion of IL2 gene expression has been
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extended by studies on the allelic expression of the IL2
genes. Under optimal TCR stimulating conditions, where
antigen is in excess, and Antigen Presenting Cells (APCs)
are not limiting, there is biallelic expression of the IL2
genes[65]. By comparison, when TCR stimulatory condi-
tions are suboptimal, then expression can be monoallelic,
and as well, fewer cells will be activated to express IL2.
However, once engaged, the rate of IL2 production per cell
remains constant. In this respect, IL2 gene expression per
cell is always quantal.
The activation of NF-AT, AP-1 and NF-κB/Rel family
members is controlled by signaling pathways triggered by
the TCR, as well as costimulatory molecules and coinhib-
itory molecules. Thus, TCR triggering promotes the rapid
influx of calcium into the cell, which activates the phos-
phatase calcineurin, thereby dephosphorylating and acti-
vating NF-AT[66], while other TCR-triggered pathways
activate the kinases p56lck, ZAP-70, PLCγ, and Protein
Kinase C-θ (PKC-θ), which simultaneously promote the
activation and translocation of AP-1 to the nucleus, and
the activation of NF-κB/Rel family members that are
already present in the cytoplasm bound to the Inhibitors
of κB (IκB)[67]. In addition, stimulation of co-stimula-
tory receptors, particularly CD28, by the B7 ligands
expressed by APCs activates PI3K, thereby further activat-
ing both AP-1 and NF-κB/Rel members [68-70].
The Phenomenon of Anergy ("Abnormal
Inactivity")
Soon after the derivation of the first T cell clones[71],
experiments with cloned helper T cells revealed that high
concentrations of specific antigenic peptide (i.e. 1–100
µM) would lead to unresponsiveness, i.e the incapacity to
produce IL2 or to proliferate when subsequently exposed
to a stimulatory concentration of antigenic peptide (i.e.1
nM-1 µM)[72]. The suppressive effect was peptide and
clone specific, took several hours to develop, and was long
lasting, up to 7 days in vitro. It could not be ascribed to
nonspecific toxicity and cell death, in that the cells were
still capable of proliferating in response to IL2 added
exogenously.
Subsequently, anergy (defined as a state of proliferative
unresponsiveness to normal mitogenic activation) was
shown to be produced by delivery of "Signal-1" (i.e. TCR),
without "Signal-2" (i.e. CD28)[11]. More recently, it has
been shown that it is possible to induce anergy pharmaco-
logically by stimulating calcium flux using calcium iono-
phores without activating the TCR or CD28[66,73,74]. In
this instance, NF-AT is activated and translocates to the
nucleus in the absence of activation of AP-1 and NF-κB/
Rel. Activation of NF-AT without the participation of AP-
1 and NF-κB/Rel results in the proteolytic degradation of
PLCγ and PKC-θ [74], as well as the transcriptional
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activation of a unique set of genes that collectively sup-
press the capacity of the cell to respond to subsequent full
TCR/costimulatory activation[73]. This induced "anergic
state" is stable and is manifest by the inability to maintain
a stable immunologic synapse, which precludes expres-
sion of the IL2 gene, so that anergic cells will not prolifer-
ate in response to a subsequent full TCR/CD28
stimulation. However, like the high antigen dose anergy,
if IL2 is supplied exogenously, the proliferative block is
bypassed, so that the anergized cells are still capable of
proliferating in response to the IL2 signals.
These findings are reinforced by other experiments, which
reveal that inhibitors of calcineurin, such as cyclosporine-
A and tacrolimus (FK506), which prevent the activation of
NF-AT, also prevent the induction of anergy[75]. As well,
T cells from NFAT-1 (-/-) mice are resistant to anergy
induction by calcium ionophores[76,77]. Moreover, NF-
AT-1 (-/-) mice exhibit a syndrome characterized by the
accumulation of hyperactivated T cells[76,77]. Thus, in
situations where calcium-mediated activation of
calcineurin and NF-AT predominates, and pairing with
AP-1 and NF-κB/Rel transcription factors does not occur,
such as in situations with little of no costimulatory activa-
tion, a biochemical milieu exists that favors the creation
of an anergic state.
By comparison, NF-κB/Rel appears to be the most critical
of the three families of transcription factors involved in
the activation of IL2 gene expression[78]. Moreover, of
the 5 members of the NF-κB/Rel family, c-Rel is the most
important, and also the critical transcription factor acti-
vated by costimulatory signals[79]. C-Rel expression is
restricted to cells of the lymphoid and myeloid lineages,
whereas the other NF-κB family members are expressed
ubiquitously in almost all tissues. T cells from c-Rel (-/-)
mice cannot express the IL2 gene or proliferate in
response to activation via full TCR/co-stimulation. How-
ever, they can proliferate normally if IL2 is supplied exog-
enously[79]. As well, c-Rel binds to the costimulatory
response element IL2 CD28RE with a high affinity (Kd =
25 nM), while NF-κB/Rel p50/p65 heterodimers bind to
this response element with a 10-fold lower affinity[80].
Accordingly, for productive CD4+ T cell activation mani-
fest by IL2 gene expression and proliferative clonal expan-
sion, the minimum requirements are optimal activation
of the TCR via peptide-MHC complexes, and costimula-
tion via activation of CD28 by the APC B7 ligands. In the
case of a low affinity TCR-MHC-peptide interaction, or in
the absence of costimulation, NF-AT activation may pre-
dominate and anergy can result[81]. The actual critical
number of TCR/CD28 activating signals that result in the
quantal expression of the IL2 gene have not been deter-
mined, but by extrapolation from the parameters regulat-
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ing IL2-IL2R activation, it is logical that peptide-MHC
ligand concentration, TCR receptor density and affinity, as
well as duration of the ligand-receptor interaction, will
dictate the number of triggered TCR/CD28 interactions,
which determine whether the cell becomes anergized vs.
activated to produce IL2, to express IL2Rs and to
proliferate.
The Regulation of IL2R Gene Expression
Resting T cells that have not been recently activated via
TCR/CD28 do not express detectable high affinity
IL2Rs[25]. A recent study carefully examined resting T
cells isolated from human peripheral blood for expression
of the 3 chains of the IL2R by flow cytometry[82]. To
ensure that the T cells represented only unactivated, truly
"resting" T cells, any in vivo activated cells were removed
using monoclonal antibodies reactive with the transferrin
receptor (CD71), known to be an early TCR/CD28 activa-
tion molecule. Both CD4+ and CD8+ resting T cells have
undetectable surface or cytoplasmic IL2R α and β chains,
as monitored using very sensitive flow cytometry meth-
ods. By comparison, IL2R γ chains are detectable in the
cytoplasm, but undetectable on the cell surface[83]. Upon
activation via the TCR/CD28, the expression of the genes
encoding both the IL2R α and β chains occurs, and γ
chains are rapidly mobilized to the cell surface, so that
high affinity trimeric IL2Rs are expressed, and the cells are
competent to respond to IL2 by proliferating.
The regulation of α chain (CD25) gene expression is
under the control of the TCR/CD28 via activation of NF-
κB/Rel, AP-1 and NFAT, which interact with 2 distinct REs
[84]. Therefore, it appears that the IL2Rα chain gene is
coordinately regulated along with the IL2 gene by the
same signaling pathways emanating from the TCR/CD28
receptors that activate the same families of transcription
factors regulating the IL2 gene. However, it has not been
determined whether the critical number of TCR/CD28
receptors necessary to trigger the IL2 gene and the IL2Rα
chain gene are similar. Most experience suggests that there
is a much lower number of triggered TCR/CD28 receptors
regulating IL2Rα gene expression as compared with IL2
gene expression, but this question needs to be examined
directly.
In addition to the TCR, IL2 enhances IL2Rα chain gene
expression as much as 10–20-fold [85-87]. This IL2 effect
on α-chain expression is readily appreciated by flow
cytometry, in that IL2 shifts the mean fluorescence inten-
sity more than an order of magnitude. As well, it is note-
worthy that the IL2Rα chain expression is ~ 10-fold higher
than expression of either the IL2Rβ or the IL2Rγ chains, so
that when using flow cytometry to detect each of the
chains, the IL2Rα chain (CD25) is always predominant.
As well, the number of functional high affinity trimeric
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IL2Rs is determined by the number of β and γc chains,
which are limiting. The energetics of IL2 chain assembly
has now provided an explanation for the excess α chains,
in that the law of mass action favors the formation of α,β
heterodimers, to which IL2 binds and then recruits the γ
chain, thereby forming the stable quaternary signaling
complex.
The In Vivo Functions of IL2
Before the identification of the IL2 molecule in
1983[57,88], it was assumed that antigens stimulate T cell
proliferation, and that mitogenic cytokines, which were
first described almost 20 years earlier[89,90], functioned
simply to amplify the signals already initiated by anti-
gen[22]. Thus, when purified IL2 became available[88]
and the IL2 receptor (IL2R) had been discovered[25],
experiments became possible for the first time to deter-
mine the molecular mechanism whereby antigen acti-
vated T cells are stimulated to proliferate. Initial studies
revealed that although antigen is necessary to activate T
cells to leave G0 and to enter early G1, cell cycle
progression through G1 to S-phase and mitosis appeared
to be mediated by IL2 upon binding to the
IL2R[26,91,92]. Resting T cells were found to be IL2R neg-
ative and IL2 unresponsive[25], while purified, homoge-
neous IL2 was capable of promoting long-term T cell
proliferation of mitogen- or antigen-activated T cells. By
comparison, mitogen or antigen alone could not sustain
long-term T cell growth[26].
Moreover, immunosuppressive pharmacological agents
such as glucocorticoids[93] were found to inhibit T cell
proliferation by preventing IL2 production but not IL2
responsiveness. As well, experiments with monoclonal
antibodies that block either IL2[88] or the IL2R[29] were
found to inhibit T cell cycle progression after mitogen or
antigen activation. These experiments all suggested that
antigen per se could not promote T cell proliferation, and
suggested that IL2 drives T cell proliferation after the ini-
tial antigen activation. Even so, the monoclonal IL2- or
IL2R-reactive antibodies suppressed proliferation by >
90–95%, but never completely abrogated proliferation,
leaving open the possibility that either the TCR itself or
other mitogenic cytokines might also be operative.
In 1991, with the advent of IL2 gene deletion through
genetic recombination, it became possible to test defini-
tively the functional importance of IL2, both in vitro and
in vivo, at least in mice. Initial in vitro experiments testing
cells from IL2 (-/-) mice for proliferation in response to
activation by the mitogenic lectin Concanavalin-A (Con-
A) revealed a 70–75% diminution of tritiated thymidine
incorporation, but not a complete abrogation[94]. There-
fore, these experiments suggested that perhaps the TCR
really was capable of promoting proliferation, and that
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IL2 functioned to merely amplify the response, as
assumed originally. Alternatively, it was also considered
possible that perhaps IL2 was not the only cytokine with
TCGF activity operative to promote T cell cycle progres-
sion, and additional cytokines such as IL4[95] or IL7[96],
which were thought originally to be primarily B cell stim-
ulators, might also be playing roles.
Even so, it was still a great surprise when initial in vivo
experiments with IL2 (-/-) mice infected with Vaccinia
Virus (VV) and Lymphocytic Choriomeningitis Virus
(LCMV), revealed that the generation of antigen-specific
effector cytolytic T cell activity was reduced by only ~ 30%
as monitored by Cytolytic T Lymphocyte (CTL) 51Cr-
release assays[97]. Moreover, neutralizing IgG antibody
responses to Vesicular Stomatitis Virus (VSV) infection, a
T-helper-dependent function, were delayed but not
reduced. Other in vivo experiments with staphylococcal
super antigens indicated that CD4+ T cells doubled nor-
mally, while CD8+ T cells from IL2 (-/-) mice were only ~
50% of wild type[98]. These findings led to the interpreta-
tion that in vivo IL2 is redundant for the generation of
immune responses, and that the TCR or other cytokines
with TCGF activity could substitute for IL2.
However, upon subsequent and more extensive testing of
IL2 (-/-) mice, it was found that in the absence of IL2, the
marked proliferative expansion of LCMV-induced CD8+ T
cells was virtually eliminated, the total cytolytic effector
capacity was reduced by > 90%, and IFN-γ production
resulting from T cell activation was dramatically inhib-
ited[99,100]. Moreover, IL2 (-/-) mice permitted pro-
longed viral replication compared with (+/+) and (+/-)
controls, which could clear the virus within a few days.
Therefore, all of these data indicated that IL2 may not be
the sole cytokine with TCGF activity, but it is one of the
principle TCGFs responsible for the maximal proliferation
of antigen selected mature peripheral T cells, as well as
their differentiation to effector cells, both in vitro and in
vivo. Moreover, IL2 (-/-) mice are immunocompromised
without IL2; thereby indicating that the TCR or other yet
undiscovered cytokines cannot fully substitute for IL2 in
vivo.
The IL2 Deficiency Autoimmune Syndrome
Since IL2 (-/-) mice are immunocompromised, it was
entirely unexpected to find that a syndrome of lym-
phocyte hyperactivity and apparent autoimmunity
appears as the mice mature beyond puberty [101]. Thus,
although lymphocyte development during embryogenesis
is grossly unperturbed by the absence of IL2, generalized
lymphoid hyperplasia ensues after the first several weeks
and months of life, and T cells that express activation
markers accumulate in the secondary lymphoid tissues. As
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well, autoimmune antibody-mediated hemolytic anemia
appears, in addition to antibodies reactive to self-mole-
cules, such as DNA and other nuclear antigens. Similar
findings with mice deleted of the IL2R α chain
(CD25)[102], β chain (CD122)[103], the JAK3 protein
kinase[104,105], and the transcription factors STAT5a/
b[106] all supported the idea that IL2 or one of the other
interleukins that signal via the IL2R γc chain [107] some-
how determines the selection of mature cells in the thy-
mus, and in the absence of postnatal IL2 expression, the
immune system begins to react to self as if it is nonself.
The accumulation of T cells with an activated phenotype
in the setting of an initial lymphopenia and immunodefi-
ciency, has recently been attributed to compensatory over
stimulation by the cytokines responsible for homeostatic
proliferation, e.g. IL7, IL15 and IL21, all of which signal
via the γc chain[108]. Of these cytokines, IL7 and IL15 sig-
nal via STAT5, whereas IL21 signals via STAT1 and STAT3.
Accordingly, IL21 stimulation via STAT1&3 may well be
responsible for the hypersensitivity lymphoproliferative
syndrome that is common to the IL2, IL2R and signaling
(-/-) phenotype.
These observations in mice were reinforced by a report of
a human homozygous mutation of CD25[109]. A male
child of first cousin parentage presented at age 6-months
with increased susceptibility to viral, bacterial, and fungal
infections, suffering from cytomegalovirus pneumonitis,
persistent oral thrush, candida esophagitis, and adenovi-
rus gastroenteritis, chronic diarrhea, and failure to thrive.
From the age of 8-months, lymphadenopathy and hepat-
osplenomegally became apparent. In vitro assays demon-
strated a reduced responsiveness to stimulation by anti-
CD3 (11% of control) and phytohemagglutinin (20% of
control). Severe immunodeficiency was proven by the
patient's inability to reject an allogeneic skin graft.
Paradoxically, despite the obvious immunodeficiency,
there was a normal sized thymus and lymphocytic infiltra-
tion of multiple tissues, including lung, liver, gut, soft tis-
sue and bone. These findings were interpreted as possibly
a result of a failure of negative selection of potential auto-
reactive cells in the thymus, as well as an inability to con-
trol autoreactive cells in the periphery, perhaps due to the
absence of CD4+CD25+ Regulatory T cells.
Regulatory T Cells (T-Regs)
Soon after it was demonstrated that IL2 (-/-) and IL2Rα or
β chain (-/-) mice develop an autoimmune syndrome, it
was reported that immunocompromised (nu/nu) mice
would also develop a wide spectrum of both organ-spe-
cific and systemic autoimmune diseases if they received
normal cell populations from which CD4+CD25+ T cells
were eliminated[110]. Furthermore, reconstitution of
CD4+CD25+ T cells in the transferred cell populations
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prevented the development of autoimmunity. Subse-
quently, we found that IL2 treatment of IL2 (-/-) mice
before day 10 after birth prevents the onset of the syn-
drome of lymphocyte activation and autoimmunity[111].
As well, thymocytes and spleen cells from IL2-treated IL2
(-/-) mice transferred to IL2 (-/-) recipients delayed the
development of the autoimmune syndrome. These data
suggested that IL2 treatment induced some normal cellu-
lar maturation/differentiation step in the transferred IL2 (-
/-) cells that subsequently prevented the cells in the IL2 (-
/-) mice from responding to self-antigens[111].
In other reports from as early as the 1960s[112], it was
established that neonatal thymectomy in the first few days
after birth can lead to subsequent widespread
autoimmune phenomena, such as hemolytic anemia, thy-
roiditis, gastritis, oophoritis, or orchitis [113-115]. These
two observations, i.e. autoimmune phenomena arising in
both IL2 (-/-) mice and neonatal thymectomized mice,
were connected when it was demonstrated that T cells
expressing the IL2Rα chain (CD25) ontogenically begin
to appear in the normal periphery immediately after day
3 of life, rapidly increasing within 2 weeks to adult levels,
which comprise ~ 10% of CD3+ cells [116]. As well, neo-
natal thymectomy on day 3 eliminates CD25+ T cells from
the periphery, and injection of CD25+ T cells from nor-
mal adult donors into day-3 neonatally thymectomized
mice prevents the development of autoimmunity, while
injection of CD25- T cells does not.
These observations were interpreted as consistent with the
notion that neonatal thymectomy on day 3 can eliminate
or reduce the autoimmune preventative CD25+ T cells,
thereby leading to unchecked activation of the self-reac-
tive T cells produced before neonatal thymectomy.
Together with the observations on the IL2 treatment of
IL2 (-/-) mice[111], these experiments fix the source of
these CD25+ cells within the thymus, and also imply that
IL2 is a necessary component in their development.
CD4+CD25+ "suppressor" regulatory T cells (T-Regs)
could also be demonstrated in the secondary lymphoid
organs of normal adult mice, and an in vitro assay was
devised to test their suppressive activity[117].
CD4+CD25+ cells typically represent ~ 10%–15% of
CD4+ T cells in lymph nodes from 8–10 week old mice.
Paradoxically in view of their expression of CD25, these
cells appear to be resting as well as anergic, in that they
cannot proliferate in response to soluble and solid-phase
anti-CD3 or Con-A. As well, even though the cells express
the IL2R α chain, they cannot proliferate in response to
exogenous IL2. However, these cells can be activated and
proliferate in response to a combination of anti-CD3 + IL2.
These data suggest that resting, anergic CD4+CD25+ T
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cells do not express the IL2R β and γ chains, but they can
be induced to express them after activation of the TCR.
When co-cultured with CD4+CD25- cells, these
CD4+CD25+ cells were found to markedly suppress the
proliferative response of the CD25- cells, provided they
are stimulated with low concentrations of soluble, but not
solid-phase anti-CD3. This inhibition was dependent on
cell-cell contact, not soluble factors, and dependent on
the suppression of IL2 production by the CD25- respond-
ing cells. The inhibition could be bypassed by the addi-
tion of IL2, or costimulation with anti-CD28. These data
were interpreted as consistent with the idea that
CD4+CD25+ cells in normal unimmunized animals rep-
resent a distinct lineage of "professional" suppressor cells
that have matured in the thymus. These T-Reg cells have
been termed "naturally occurring" (nT-Reg).
It has also been reported that CD4+ T cells with regulatory
function can be generated in vitro by the activation of
mature peripheral CD4+CD25- T cells[118]. Thus, both
regulatory and effector cells can, in principle, be generated
from the same mature CD4+ T cell precursors. It has been
postulated that these "inducible" cells (iT-Regs) might be
triggered by "low-affinity or altered TCR signal
transduction"[118], in that the conditions that favor the
generation of T-Regs ex vivo from mature CD4+CD25- T
cells, include antigen in the presence of immunosuppres-
sive cytokines such as IL10 and TGFβ, immunosuppres-
sive agents such as vitamin D3 and dexamethasone,
CD40-CD40L blockade or immature DC
populations[119,120].
Furthermore, it was reported recently that subcutaneous
infusion of low doses of antigenic peptide by means of
osmotic pumps over 14 days transforms mature periph-
eral T cells into CD4+CD25+ "suppressor cells" that can
persist for long periods of time (i.e. several months) in the
absence of antigen and confer specific immunological
tolerance upon challenge with immunogenic doses of
antigen[121]. Therefore, it appears that both in vitro and
in vivo, low antigen concentrations can promote the differ-
entiation of mature peripheral CD4+ T cells to express
CD25, and to become "suppressor" cells rather than effec-
tor cells.
The dependency on the thymus for maturation of
CD4+CD25+ T-Regs, as well as the dependency upon IL2,
has been underscored by experiments with IL2β chain (-/
-) mice made transgenic for the IL2Rβ chain under the
influence of the proximal lck promoter, so that mature
trimeric IL2Rs capable of signaling are only expressed in
the thymus[122,123]. Transgenic expression of the IL2Rβ
chain in IL2Rβ chain (-/-) thymocytes corrects the lack of
CD4+CD25+ peripheral T cells in IL2Rβ chain (-/-) mice
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and prevents lethal autoimmunity. These experiments fur-
ther emphasize the unique contribution of IL2 to the
development of CD4+CD25+ T-Regs, in that the other γc
chain cytokines, e.g. IL4 and IL7, IL9, and IL21 do not
compensate for the lack of T-Regs in IL2Rβ chain (-/-)
mice.
Suboptimal antigen concentrations and IL2 are both
required for the generation and activity of T-Regs, and
both in the thymus and in the periphery. However, the
forces governing the simple generation of anergic and
nonfunctional CD4+CD25+ T cells, as compared with the
determinants of the generation of activated proliferating,
suppressive CD4+CD25+ "Regulatory T cells", vs.
CD4+CD25+ activated proliferating functional effector T
cells remain to be defined.
Because each of these cell fates is functionally distinct, it is
reasonable to hypothesize that these distinct cell fates are
determined by different numbers of triggered TCRs and
IL2Rs.
The Transient Nature of the In Vitro T Cell
Proliferative Response: Feedback Inhibition of
IL2 Gene Expression
Upon productive activation of T cells in vitro via TCR/
CD28, there is a characteristic transient expression of the
IL2 gene, such that IL2 mRNA first becomes detectable
within 6 hours, and then peak levels occur after 12 hours,
with a subsequent decline to undetectable levels by 24
hours[21]. Detectable IL2 protein in the culture media fol-
lows a similar, but delayed course with peak concentra-
tions found after 24 hours, and by 48 hours barely
detectable levels remain. Expression of high affinity
trimeric IL2Rs follow a similar, but delayed transient
expression course, with peak levels expressed at 24–48
hours, followed by a slow decline over several days[91].
By comparison, expression of the IFN-γ gene follows a
much more protracted course, with detectable expression
still evident after several days[124].
The mechanisms accounting for the transient expression
of the IL2 gene and the genes encoding the IL2R chains
have not been apparent, until recently. It is now realized
that surface molecules of the coinhibitory CTLA-4 fam-
ily[125] appear later after TCR/CD28 activation, first evi-
dent after ~ 24 hours with peak levels at 48–96 hours. In
addition to CTLA-4, which has a 10-fold higher affinity for
the B7 molecules expressed by APCs, the coinhibitory
receptor PD-1[126], as well as the ligands reactive with
this receptor, PDL-1[126] and PDL-2[127], appear on
activated T cells. Still a third, later appearing coinhibitory
receptor, BTLA, has also recently been described as
expressed on both antigen-activated T cells and B
cells[128].
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This coinhibitory ligand-receptor family of molecules
belongs to the Ig superfamily and the B7/CD28 costimu-
latory ligand/receptor family[129,130]. However, the
CTLA-4 family of receptors does not function to deliver a
costimulatory signal, as does CD28. Instead, members of
the CTLA-4 family have inhibitory signaling motifs in
their cytoplasmic domains and they have been shown to
localize in close proximity to CD28, where they compete
with activating signals from CD28. For example, by bind-
ing the phosphatase SHP-2, the CTLA-4 family of mole-
cules inhibits the positive signals emanating from the
TCR/CD28 at a very proximal position within the signal-
ing cascade, thereby extinguishing the expression of the
IL2 gene. Once IL2 production is shut down, because IL2
is internalized and degraded with a half-time (t1/2) of ~ 2
hours, IL2 is consumed very rapidly, resulting in cessation
of IL2-promoted cell cycle progression and eventually
apoptosis due to the lack of IL2-promoted anti-apoptosis
gene expression, such as Bcl-XL.
The coinhibitory ligand/receptor pairs identified thus far
can be shown to exert their negative effects by attenuating
IL2 gene expression, but the administration of IL2 can
bypass this block, in that the cells are still IL2-responsive.
A similar phenomenon has been found with regard to the
effects of T-Regs. T-Regs shut down IL2 gene expression,
via a cell-cell contact mechanism that has not yet been
delineated. However, IL2 supplementation will bypass
the suppressive effects of T-Regs, thereby allowing for T
cell proliferation. Also, in both instances, activation of
CD28 via monoclonal antibodies serves to counteract the
blockades, and permits the suppressed cells to both
express the IL2 gene and to proliferate.
Accordingly, it would seem a plausible hypothesis that the
cell-cell contact mechanisms employed by T-Regs to
inhibit IL2 gene expression are mediated by members of
the CTLA-4 family of coinhibitory ligands/receptors,
either those already identified, or others yet to be identi-
fied. This is a particularly attractive hypothesis, in that the
ligands that trigger the coinhibitory PD-1 receptor are also
expressed by TCR/CD28-activated T cells[131].
The Transient Nature of the In Vivo T Cell
Proliferative Response And "Adaptive
Tolerance"
With the advent of the ability to label cells with 5- and 6-
carboxyfluorescein diacetate succinimidyl ester (CFSE),
combined with the use of gene deleted mice, it has been
possible to follow the proliferation of T cells in vivo, and
to test which proliferative signals are operative. In this
regard, T cells from IL2Rβ (-/-) mice that have had the
IL2Rβ chain expressed only during thymopoiesis do not
express detectable IL2Rβ chains in the periphery, and
therefore are incapable of delivering a proliferative signal.
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However, these cells are capable of undergoing 1–2 divi-
sions upon stimulation with anti-CD3 + anti-CD28[132].
Similar findings have been reported in systems using TCR
transgenic mice and adoptive transfer experiments when
either IL2 or CD25 have been deleted[133,134]. One
interpretation is that TCR/CD28 activation may be capa-
ble of initiating 1–2 rounds of cell division, but IL2
appears necessary for maximal and sustained prolifera-
tion. Alternatively, other, yet undiscovered cytokines with
TCGF activity may be responsible for activating the initial
proliferative responses to antigen activation.
After the initial proliferative clonal expansion of antigen-
selected cells, the fate of the expanded effector cells has
been found to depend greatly upon whether the antigen is
cleared or whether it persists. In experimental viral infec-
tions where the virus is cleared rapidly within the first
week after infection, expanded CD4+ and CD8+ effector
cell populations undergo a contraction, with the loss of as
much as 90% of the expanded effector cells[18]. We have
found that this contractive phase is attributable to
cytokine withdrawal apoptosis, in that the administration
of IL2 during this phase prevents the contraction[135].
Others have shown a similar protective effect of IL2 after
both CD4+ and CD8+ cells are expanded in response to
activation by staphylococcal superantigen[136]. Subse-
quent studies have revealed that the residual populations
of expanded effector cells eventually differentiate to "cen-
tral" memory cells, which have the capacity for mainte-
nance of the population size via slow proliferative renewal
and as well, the capacity to respond to the reintroduction
of antigen by rapidly producing IL2, proliferating and dif-
ferentiating to effector cells[137].
With persistence of antigen, the fate of the expanded effec-
tor T cell populations changes dramatically. Instead of dif-
ferentiating into responsive memory cells, the cells revert
to a state of unresponsiveness, which has been termed
"exhaustion" by those studying experimental persistent
viral infections [138,139]. This exhausted state is mani-
fested by an early loss of the capacity to produce IL2 and
to proliferate. As antigen persists, the cells gradually lose
their capacity to lyse target cells and to secrete antiviral
effector cytokines such as TNF-α and IFN-γ. Eventually,
clones of virus-specific cells can undergo apoptosis, which
may be attributed to Activation-Induced Cell Death
(AICD), leading to clonal disappearance[140].
A similar phenomenon has been described in experiments
employing a paired transgenic model (TCR and Ag)[141].
In this model, CD4+ TCR-Tg T cells from antigen-naïve
animals are labeled in vitro with CFSE, then transferred
into recipient mice expressing low levels (~ 100 pM) of
antigenic pigeon cytochrome c peptide. The cells become
activated, express the IL2R α-chain (CD25) and CD69,
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and proliferate for the first 4 days, eventually expanding ~
100-fold. However, over the subsequent 10–14 days, the
cells lose expression of CD25 and cell recovery declines by
~ 50%. Thereafter, the cells are said to be in the "adaptive
phase", which is characterized by a hyporesponsiveness to
antigen in vitro, and is manifest by a decreased capacity to
produce IL2 and other cytokines, and to proliferate.
This adaptive state persists in the host with chronic expres-
sion of the antigen, and in contrast to a similar paired
transgenic model in a B cell system[142], is not associated
with a decrease in the level of expression of the TCR[143].
However, the adaptive state is dependent upon continu-
ous exposure to antigen; upon transfer to an antigen-neg-
ative host, the hyporesponsive state reverts. Moreover, the
adaptive state is similar to a desensitization phenomenon
akin to tachyphylaxis, and studies have revealed a down
regulation of the TCR signaling molecules, involving an
early block in tyrosine kinase activation, which primarily
inhibits calcium mobilization, thereby suggesting that the
desensitization involves the adaptation of the TCR signal-
ing apparatus to the chronic persistence of low levels of
antigen[143,144].
It is important to emphasize that the adaptive state is only
a relative hyporesponsiveness, as compared to either the
naïve situation or the host with central memory
cells[141,143]. If cells are exposed to higher concentra-
tions of peptide in vitro, i.e. between 1 nM and 1 µM, a
response can be detected, but the antigen dose-response
curve is shifted 100–300-fold to the right. In addition, the
adaptive phenomenon cannot be ascribed to active sup-
pression by a T-Reg differentiative process, in that the
adapted cells do not express CD25, and in vivo experi-
ments have excluded a suppressive mechanism.
What Determines the "Strength" of the Signal?
From the discussion thus far, it must be apparent that the
strength of the signals delivered to T cells ultimately deter-
mines the outcome, i.e. either anergy, or activation of the
IL2 gene, and if activation occurs, the duration that it per-
sists. Thus, considerations of the ligand concentrations
available, the receptor affinities and numbers expressed,
and the duration of the ligand-receptor interactions again
become important.
The duration of signaling via TCR/CD28 is known to be a
major determinant of the magnitude of IL2 production
and thus the extent of T cell proliferation. Even in the
1970s the magnitude of the proliferative response after
mitogenic lectin administration was found to be directly
related to the duration of lectin stimulation[145]. Thus,
removal of the mitogenic lectin within the first 24 hours
of stimulation attenuates the proliferative response
markedly.
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With the discovery that T cell proliferation after mitogen
or antigen activation is principally mediated by IL2[22], it
seemed obvious that a certain time interval was necessary
after TCR/CD28 triggering to provide for maximal expres-
sion of the IL2 and IL2R genes. Indeed, experiments
proved this to be the case[146]. Even so, several hours
seemed somewhat prolonged, given that early biochemi-
cal events such as calcium flux and kinase activation were
detectable within minutes after TCR engagement. Moreo-
ver, IL2 gene transcription could be detected within 45
minutes, using sensitive techniques[147].
A series of experiments reported in the mid 1990s began
to provide an explanation for this perplexing problem. By
equating "triggered" TCRs with their internalization and
disappearance from the cell surface after ligand activation,
it was shown that a single peptide-MHC complex is capa-
ble of serially triggering up to ~ 200 TCRs[148,149]. Fur-
thermore, like the IL2Rs, T cells appeared to be able to
"count" the number of triggered TCRs, and responded by
proliferating when ~ 8,000 TCRs were triggered[149,150].
Other experiments showed that the duration of antigenic
stimulation was one of the most critical parameters deter-
mining the fate of naïve and effector T cells, i.e. whether
they would be activated or deleted[151]. However, these
observations were not linked to the IL2/IL2R system.
With the discovery of Supramolecular Activation Clusters
(SMACs), an additional level of complexity was
added[152]. Also termed the "Immunological Synapse",
the specialized junction between a T cell and an APC con-
sists of a central cluster of T cell receptors together with
costimulatory and coinhibitory receptors, surrounded by
a ring of adhesion molecules[153]. Recent experiments,
which employed peptide antigen-specific TCR αβ
transgenic T cell blasts labeled with CFSE, the relationship
between the duration of the TCR signal and the extent of
the proliferative response could be examined further at
the single cell level. Between 10–24 hours of continuous
TCR stimulation by MHC-peptide in a stable SMAC is nec-
essary to promote maximal IL2 production and
proliferation[154].
Regarding the density of the TCR on responding T cells, it
is important to restate and emphasize that the TCR den-
sity follows the same log-normal distribution as detailed
for the log-normal distribution of IL2Rs, even on cloned T
cells. Thus, there is at least a 2-log10 difference in TCR den-
sity among potential responding T cells, and conse-
quently, those cells with the highest density of TCRs, will
be capable of responding to lower concentrations of
MHC-peptide epitopes, and also capable of responding
more rapidly than cells with lower TCR densities to an
optimal pMHC concentration. Detailed studies examin-
ing the cytokine response of murine T cell clones to
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graded peptide antigen concentrations have revealed a
hierarchical organization of TCR signal-dependent
response thresholds for elicitation of different cytokines
in individual cells[155]. IL2 production was found to
remain constant per cell as the ligand concentration was
increased, with the primary change being in the number
of cells making IL2 at a fixed level. Therefore, the decision
to produce IL2 is a quantal decision on the part of each
individual cell within the cloned population.
Exactly what leads to the heterogeneity of the quantal
response of IL2 gene expression on the part of the cells
within the cloned cell population has not been deter-
mined, but very similar findings have been reported for
IFN-γ production by a human T cell clone in response to
graded doses of antigenic peptide[156]. The peptide dose
that stimulated 5% vs. 95% of the T cells was found to
span over a log, and the response on the part of the cells
comprising the population was quantal; i.e. at low antigen
concentrations fewer cells expressed IFN-γ and as the anti-
gen concentration was increased, an increasing number of
cells expressed IFN-γ. To explain these results it was postu-
lated that the intraclonal heterogeneity in antigen respon-
siveness could result from the different numbers of TCRs
expressed by individual cells. However, this conjecture
has not been examined directly.
Exactly the same considerations hold for the distribution
of costimulatory and coinhibitory molecules. Thus, there
is interplay between all of these receptors, which ulti-
mately impacts the "strength" of the signal[157] and the
duration that the signal must be applied to productively
signal the IL2 gene response elements, and eventually lead
to an "activated" T cell. Obviously, if any of these param-
eters are limiting, the activation events may favor the
delivery of abortive transcriptional activating signals,
which may favor anergy or differentiation to T-Regs,
rather than activation[141].
As already detailed, if CD4+ T cells are activated via the
TCR without adequate stimulation via CD28, such as may
occur if self peptide is presented on immature dendritic
cells as APCs, the conditions would favor the
predominant activation of NF-AT yet inadequate AP-1
and NF-κB/Rel activation, which would promote anergy
and perhaps even the irreversible differentiation of most
of the cells to T-Regs. Thus, the concentration of antigen,
the availability of adequate costimulatory molecule func-
tion, the affinity and density of the TCR/cell, as well as the
duration that the TCR is triggered all influence signal
"strength".
With regard to the affinity of the TCRs for the peptide-
MHC complex, it is important to note that the TCR does
not undergo somatic hypermutation and "affinity matura-
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tion" as does the BCR. Accordingly, the Kd of the TCR is
fixed after recombination and rearrangement in the thy-
mus, and is relatively low by comparison with the Kd of
antibody molecules, which have ~ 1000-fold higher affin-
ity for binding antigen. Measurements of the equilibrium
dissociation constants of isolated agonist pMHC binding
to TCR molecules by surface plasmon resonance have
revealed Kds in the range of 1–100 µM, with koff rate con-
stants of ~ 0.01–0.1 s-1, yielding t1/2 ~ 7–70 secs[158]. In
contrast, antagonistic MHC-peptide-TCR interactions off-
rates are ~ 10-fold faster than agonistic interactions. Thus,
off-rate constants of ~ 5 sec-1 have been found, which yield
t1/2 of only ~ 0.15 seconds.
Of course, the TCR does not bind MHC-peptide com-
plexes in isolation or in solution. The formation of the
immunological synapse greatly alters the way in which
TCRs engage antigens and the way in which they are trig-
gered[55]. Thus, T cell clones that have TCRs with a Kd =
1 µM binding to MHC-peptide in isolation can be trig-
gered at peptide concentrations ranging as much as 100-
fold lower in vivo. Furthermore, new studies have made it
possible to "count" the exact number of ligands that a T
cell encounters on another cell, and then monitor the con-
sequences of that interaction with respect to the increase
of intracellular calcium concentration[55]. It has been
found that only 10 MHC-peptide ligands are sufficient to
provide for the formation of a stable immunological syn-
apse and sustained calcium flux for several hours. How-
ever, below this critical number of MHC-peptide ligands,
only transient calcium increases occur, and a stable syn-
apse does not form. Thus, an abortive signaling process
appears to ensue, which could very well lead to the activa-
tion of NF-AT without adequate levels of AP-1 or NF-κB/
Rel, which would be insufficient for IL2 and IL2Rα chain
gene expression, thereby promoting anergy/T-Reg
differentiation.
Recent experiments focused on how the TCR can respond
to such low concentrations of agonist peptides indicate
that the slower off rate of the agonist pMHC/TCR interac-
tion allows the juxtaposition of CD4 with bound lck to the
agonist pMHC/TCR, so that endogenous pMHC/TCR,
which are in a large excess, can form a dimeric signaling
complex comprised of agonist pMHC and endogenous
pMHC[159]. Then, the endogenous pMHC with its fast
off rate can trigger many TCRs serially and greatly amplify
the TCR signals.
Given the long duration necessary to trigger a response, a
kinetic model has been proposed to account for how seri-
ally triggered TCRs that interact very briefly with peptide-
MHC complexes, then are rapidly internalized and
degraded can be counted by the T cell, and how transient
signaling events can be accumulated over time and
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Medical Immunology 2004, 3:3
integrated into a quantal response[160]. The model is
based on a process first described in neuronal cell activa-
tion termed 'temporal summation'. The signaling events
originating from successively triggered TCRs build up,
with each adding to the falling phase of the one before. In
this way, small and short signals that alone are unable to
trigger a response can be summed up over time eventually
to reach the level sufficient to trigger the quantal response,
in this instance IL2/IL2R gene expression.
All of these considerations lead one to the conclusion that
like the IL2/IL2R-determined quantal decision to undergo
cell cycle progression, there appear to be quantal deci-
sions operative at the level of the immunological synapse
that lead to distinct cell fates, which in turn are ultimately
determined by IL2 and IL2R gene expression. Thus, if the
agonist pMHC ligand concentration is low, only T cells
with a high density of TCRs will form stable synapses that
will result in sustained activation of the IL2 gene and
thereby cell cycle progression. It follows that at the same
limiting agonist pMHC concentrations, cells with lower
TCR densities may have abortive expression of the IL2
gene, which would favor differentiation to T-Regs, while
cells with still lower TCR densities would not successfully
trigger expression of the IL2 gene, thereby favoring the
triggering of differentiation to an anergic state and unre-
sponsiveness. Thus, there are at least 3 distinct cell fates
that are determined by the accumulated number of trig-
gered TCRs, which is determined by the agonist pMHC
concentration, TCR density and the duration of the
pMHC/TCR interaction.
The Quantal Numbers of Triggered Receptors
are Specified in the Thymus
The structure and function of the immunological synapse
essentially determines the fate of T cells as they mature in
the thymus. Again, this cell fate determination is linked to
IL2 and IL2R gene expression. From the above discussion,
it is now clear that like the quaternary IL2/IL2R complex,
the immunological synapse is a dynamic multicompo-
nent molecular complex, the stability of which requires
ongoing signaling through the TCR for stable calcium
mobilization and kinase activation occurring over several
hours. As well, the immunological synapse modulates the
overall level of mature T cell activation by integrating pos-
itive (costimulatory) signals and negative (coinhibitory)
signals from a variety of surface receptors. In this regard, it
is noteworthy that the synapse that forms between thymo-
cytes and thymic stromal cells differs qualitatively from
that observed between mature peripheral T cells and
peripheral APCs[161]. One reason that this may occur
relates to the lack of expression of the costimulatory B7
molecules on thymic stromal cells[162]. In this regard, on
would predict that thymocytes would not be activated to
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produce IL2 very readily, due to the lack of CD28/B7
costimulation.
From detailed experiments performed primarily with
mice made transgenic for the TCR, it is now clear that the
interaction of the TCR expressed on developing immature
thymocytes with self peptide-MHC molecules expressed
on thymic stromal cells is essential for the selection of
those cells that ultimately are destined to leave the thymus
and populate the periphery [163-166]. Thus, after produc-
tive rearrangement and expression of the αβ chains of the
TCR, four fates are possible. If there is little or no affinity
of the TCR for self peptide-MHC molecules, the T cells
undergo apoptosis as a result of a lack of signal genera-
tion. However, if there is a "weak interaction" between the
TCR and nonagonist or antagonist self peptide-MHC mol-
ecules (i.e. those molecules that have a rapid off-rate from
binding to the TCR), the maturing T cells are "positively
selected" to survive. Although it still remains controver-
sial, most data are consistent with the notion that an inter-
mediate strength of signal leads to the differentiation of T-
Regs. By comparison, if the αβ TCR encounters an agonis-
tic self peptide-MHC interaction, i.e. one that has a slower
off-rate and a higher affinity, "negative selection" occurs
and these T cells are induced to undergo apoptosis. Con-
sequently, only those T cells that have "nonagonistic"
reactivity with self peptide-MHC molecules make up the T
cell repertoire.
With regard to the generation of quantal cellular
responses, it is noteworthy that IL2 has now been impli-
cated to be involved in both positive and negative selec-
tion, as well as T-Reg differentiation.
Recent experiments focused on the signals generated in
thymocytes leading to positive selection have revealed
that signaling via calcium and calcineurin is necessary for
positive selection but dispensable for negative selec-
tion[167]. For example, deletion of the regulatory subu-
nit-B1 of calineurin in thymocytes leads to loss of
activation of NF-ATc proteins and also inefficient ERK
activation, but normal activation of NF-κB/Rel. Of inter-
est, positive selection was found to be markedly deficient
in these animals, but negative selection remained intact.
As already discussed, experiments performed with IL2,
IL2R, JAK3, and STAT5 (-/-) mice have all now demon-
strated that the IL2/IL2R interaction is unnecessary for
positive selection. In the absence of signals generated via
the IL2/IL2R interaction, positive selection proceeds
unimpeded, so that during development and after birth, a
normal number and composition of cells mature in the
thymus and populate the peripheral lymphoid tissues.
Thus, if the "weak" signals between the TCR and self pep-
tide-MHC are not strong enough to trigger expression of
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Medical Immunology 2004, 3:3
the IL2 and IL2R genes, the cells are permitted to survive,
and leave the thymus to populate the secondary lymphoid
tissues. Should agonistic nonself-peptide-MHC
complexes be introduced that are capable of binding to
the TCR with high affinity, these T cells, which are non-
reactive with "nonagonistic" self peptide-MHC com-
plexes, are not anergic. Rather, if stimulated by non-self
agonistic peptide, they are fully capable of expressing IL2
and IL2R genes, and of undergoing IL2-dependent prolif-
erative expansion and differentiation to effector cells in
the periphery.
However, the development of T-Regs (i.e. CD4+CD25+
cells) is dependent on the IL2/IL2R interaction. In the
absence of IL2 or functional IL2Rs, T-Regs are low or
absent, both in the thymus and in the periphery. Moreo-
ver, if the IL2/IL2R interaction is restored, either geneti-
cally or pharmacologically, then T-Regs are reconstituted
and the autoimmune phenomena are delayed or pre-
vented altogether[106,111,168-170]. As well, the func-
tion of T-Regs in the periphery is also totally dependent
on IL2/IL2R signaling, so that if potential positively
selected autoreactive T cells are not continuously sup-
pressed by T-Regs, the IL2 (-/-) syndrome of lymphoid
hyperplasia and autoimmunity will occur.
At this juncture, it is logical to propose that the number of
triggered TCRs and IL2Rs receptors necessary to generate
T-Regs must be higher than the number required to gener-
ate simple "positive selection, in that IL2/IL2R gene
expression must be triggered[73,171]. In this regard, it has
been reported that α chain allelic "inclusion" results in a
lower density of TCRs/cell, in that the β chains are paired
with 2 distinct α chains in ~ 30% of αβ TCRs[172]. Upon
introduction of antigenic peptide, these TCR bi-allelic
cells can escape negative selection, presumably because
their epitope-specific TCR density is only half normal, and
accordingly they would not accumulate the same number
of triggered TCRs as a mono-allelic cell. This difference
could be responsible for the generation of T-Regs.
It also appears that differentiative signals triggered by the
IL2/IL2R interaction are necessary to promote the differ-
entiation to an anergic and suppressive T-Reg cell[81].
Furthermore, the number of triggered TCR receptors must
be lower than those necessary to trigger "negative selec-
tion". These findings have led some investigators to pro-
pose that the main "nonredundant" function of IL2 is to
promote the development and function of T-
Regs[173,174].
"Negative selection" of potential self-reactive T cells has
been proposed to occur via a TCR-triggered apoptosis. The
exact molecular mechanisms responsible for this effect
still remain obscure, but apoptosis appears to occur when
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there is a strong agonistic TCR-self-peptide-MHC interac-
tion, which should trigger maximal IL2 and IL2R gene
expression. However, data have been reported indicating
that negative selection of CD8+ T cells proceeds normally
without IL2[175]. By comparison, it remains controver-
sial whether the IL2/IL2R interaction is necessary for neg-
ative selection of CD4+ T cells[176].
Quite convincing data have revealed a role for IL2 in
CD4+ T cell negative selection [177] using nontransgenic
and transgenic IL2-sufficient and deficient animal model
systems. It could be shown that during TCR-mediated thy-
mocyte apoptosis, IL2 protein is expressed and detectable
in situ in the thymus, and apoptotic thymocytes up-regu-
late the expression of IL2Rs. Furthermore, IL2R+ CD4CD8
double-positive and CD4 single-positive thymocytes
undergoing apoptosis bind and internalize IL2. As well,
IL2-deficient thymocytes are resistant to TCR/CD3-medi-
ated apoptotic death, which is overcome by providing
exogenous IL2 to IL2 (-/-) mice. Finally, disruption or
blockade of IL2/IL2R interactions in vivo during antigen-
mediated negative selection rescues MHC class II
restricted thymocytes from apoptosis. Thus, all of these
findings provide evidence for the direct involvement of
the IL2/IL2R signaling pathway in the deletion of self-
reactive double-positive and CD4 single-positive T
cells[177].
Accordingly, these data are all entirely consistent with the
notion that the CD4+ T cell hyperplasia and autoimmu-
nity observed in IL2 (-/-), IL2R (-/-), and IL2 signaling (-/
-) mice are attributable, at least in part, to inefficient dele-
tion of strongly agonistic self-reactive CD4+ T cells, as well
as deficient maturation of T-Regs.
How Can the Immune System Ever
Discriminate Between "Self & Non-Self"
Peptides?
The foregoing considerations lead one to the realization
that there are no known molecular mechanisms that can
explain how the TCR can discriminate qualitatively
between peptides of self-origin vs. peptides of nonself-ori-
gin. Both of these ligands are identical in structure, i.e.
they are both peptides. Moreover, the αβ TCRs are also
identical structures, whether they recognize self or non-
self peptides bound to MHC. It follows that all of the data
and logic support a quantitative mechanism of discrimina-
tion based upon the accumulated number of triggered
TCRs and IL2Rs, as shown in Figure 6. Moreover, each trig-
gered cellular differentiative fate of survival, death,
anergy, or proliferative expansion, is quantal.
Both in the thymus and in the periphery, there are 3 cellu-
lar fates specified by an increasing number of triggered
TCRs, which dictates whether IL2 is produced and how
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Figure
The number
6 of triggered TCRs and IL2Rs determine quantal T cell fates in both the thymus and the periphery
The number of triggered TCRs and IL2Rs determine quantal T cell fates in both the thymus and the periphery. On each plot,
the number of triggered TCRs and IL2Rs increase from bottom to top. The different quantal cell fates are dictated by a definite
number of triggered Rs as depicted.

much IL2 is produced. Thus, ultimately, the number of
IL2-triggered IL2Rs determines the critical quantal fate
decisions. A similar conclusion was introduced recently,
with the difference that TCR avidity (i.e TCR affinity ×
density) was postulated to dictate the cell fates, but no
role was postulated for IL2[178]. Since ultimately, self-
nonself discrimination of the immune system depends on
proliferative expansion of antigen-selected clones, the
connection between the number of triggered TCRs and
IL2Rs offers a molecular explanation for the quantal cellu-
lar response.
If T cells that have potential reactivity with self peptide-
MHC ligands exit the thymus having escaped negative
selection, these T cells will populate the secondary lym-
phoid tissues in the periphery at very low frequencies, ~ 1
in a million lymphocytes. Thus, these cells make up the
TCR repertoire in the periphery, and as long as the distinct
self-pMHC complexes remain below the critical number
necessary for the formation of a stable synapse, and the
TCR-pMHC off-rate is rapid, there is no necessity to
introduce any additional mechanism to allow the
immune system to ignore self. Instead, in the periphery
the immune system only recognizes peptides, whether self
or non-self, which are present at a high enough concentra-
tion to attain the critical density of 10 peptide-MHC mol-
ecules/synapse and as well, that generate a slow enough
off-rate from the TCR to form a stable synapse, so that the
critical number of triggered TCR/CD28 receptors for acti-
vation is reached. The only caveat beyond these consider-
ations is that if an abortive pMHC/TCR synapse forms, the
cell receives signals that it interprets as instructions to
become anergic, or possibly to differentiate to become
suppressive cells (T-Regs), thereby solidifying the non-
reactivity on the part of the host to these peptides.
Conclusions
The "Quantal Theory" states that the fundamental deci-
sions of the T cell immune system are dependent upon the
cells receiving a critical number of triggered TCRs and

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Medical Immunology 2004, 3:3
IL2Rs and that the cells respond in an all-or-none fashion.
Any reductionist approach to understand how the
immune system discriminates self from non-self must
begin with a systemic immunological response that most
closely correlates with immunity. Thus, the "Quantal
Theory" is based on Burnet's axiom that the proliferative
expansion of antigen-selected clones is central to the gen-
eration of a protective immune response[1]. Secondly, a
successful reductionist theory must explain how individ-
ual cells of the immune system make the decision to pro-
liferate or not. As each decision of the individual cell is
quantal, one must explain the molecular basis of the
quantal cellular decision. At each decision point, the
assembly of essentially irreversible multicomponent mac-
romolecular complexes underlies the quantal cellular
responses. Given the data that the simple IL2/IL2R inter-
action promotes the quantal decision to undergo cell cycle
progression by reaching a critical number of triggered
IL2Rs[26], it follows that the quantal decision to express
the IL2 gene and the IL2R genes is similarly regulated by a
critical number of triggered TCR/CD28 molecules. Thus,
the TCR/CD28-triggered expression of the IL2 and IL2R
genes is pivotal for the quantal cellular decisions in the
thymus that determine distinct fates such as positive selec-
tion (survival), the differentiation to T-Regs (anergy), and
negative selection (apoptosis), while it is also pivotal for
the quantal cellular decisions in the periphery that
determine whether to remain unresponsive (survival), to
differentiate to T-Regs (anergy), or to begin proliferating
(immunity). Accordingly, the "Quantal Theory" offers a
unifying explanation at the molecular level that provides
the cellular mechanisms for the immune system as a
whole to make the quantal discrimination between self
antigens and nonself antigens.
These considerations lead to the speculation that non-self
peptides that are introduced in low enough concentra-
tions may well be perceived by the immune system as
"self" and will generate tolerance. Thus, we now have a
molecular, cellular and immunological explanation for
the phenomenon of "Low Zone Tolerance", first demon-
strated by Mitchison 40 years ago[16]. Accordingly, one
might have a means to tolerize individuals to specific
defined peptides that may be useful in the treatment of
allergy, autoimmunity, and allograft rejection. In contrast,
a similar situation could be operative in the tumor-bear-
ing host, and in the host infected chronically with viruses
such as the Human Immunodeficiency Virus, and Hepati-
tis C Virus. In these instances, low persistent antigen levels
may well serve to maintain a state of "low zone" tolerance.
Accordingly, the question arises as how to break this tol-
erant state?
Competing interests
The author declares that he has no competing interests.
http://www.medimmunol.com/content/3/1/3
Acknowledgements
The author wishes to thank Drs. Marc Feldmann, Ellis Reinherz, Sofija And-
jelic, Hsiou-Chi Liou, Zoran Popmihajlov and Anjana Rao for reading the
manuscript and for their helpful comments and suggestions. The author is
also thankful for financial support from the NIAID, NIH (Grants R01-AI
44207, U01-48224, R01-51181) the Weill Cornell General Clinical
Research Center (Grant M01-00047) and The Doris Duke Charitable
Foundation.
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